We compared the adsorption and desorption of 1-pyrenecarboxylic acid (PyC), 1-pyreneacetic acid (PyA),and 1-pyrenebutyric acid (PyB) on silver nanoparticles (AgNPs) via interfacial spectroscopic tools to studythe role of the aliphatic units between pyrene and carboxylic group. The negative surface charges ofAgNPs at ca. −51 mV shifted up to −11 mV, after adsorbing hydrophobic pyrene compounds. The threepyrene acid compounds appeared to adsorb onto AgNPs via their carboxylate units by referring to theobservation of the broad (COO−) bands at 1380–1410 cm−1in the Raman spectra. X-ray photoelectronspectroscopy (XPS) also supported the C O species in the C1sregion. AgNPs were found to efficientlyquench the fluorescence of the three pyrene acid adsorbates. The highest Stern–Volmer constant of PyAmay be due to the largest overlap integral with the surface Plasmon absorption band of AgNPs. The butyricunit was expected to lead a stronger binding on Ag, as suggested by density functional theory (DFT) cal-culations. The adsorbed pyrene acid compounds appeared to be released from AgNPs by thiol-containingglutathione (GSH). PyB with the butyric group exhibited larger quenched fluorescence intensities andsmaller released amounts than PyC and PyA in aqueous solutions and A549 cancer cells. Our study willbe helpful in designing pyrene-based fluorescence sensors in cellular imaging.